Reagents for the determination of cerebral regional acetylcholinesterase activity

ABSTRACT

The present invention relates to N-alkylpiperidine derivatives represented by general formula (1) or (2);  
                 
 
     wherein R 1  represents optionally fluorinated lower alkyl; R 2  represents lower alkyl; and R 3  represents alkenyl substituted at the 1-position with hydroxy, lower alkoxy, lower alkoxyalkyloxy, lower alkoxyalkyloxyalkyloxy, or lower alkanoyloxy and substituted at the end with radioactive iodine, or alkenyloxymethyl substituted at the end with a radioactive iodine reagent containing the same for assaying central local AchE activity; a method for assaying the central local AchE activity; and labeled precursors of the above compounds. After easily passing through the blood-brain barrier, these compounds are hydrolyzed specifically by AchE in the brain into alcohols, which are then captured by the brain. In contrast, alcohols formed outside the brain do not migrate into the brain. The compounds of the invention emit γ-rays at an appropriate energy level. These characteristics make the compounds highly useful as tracers for SPECT in assaying the central AchE activity.

TECHNICAL FIELD

[0001] The present invention relates to N-alkylpiperidine derivativesand reagents including the derivatives used for the determination ofcerebral regional acetylcholinesterase (AchE) activity.

BACKGROUND ART

[0002] Cerebral cholinergic nerve system plays an important role inmemory function. Degeneration of this nerve system is thought to beimplicated in memory impairment seen in dementing disorders such asAlzheimeer's disease. The AchE activity has been found to be reduced inaccordance with the decreased cholinergic function in the brain. Thedetermination of cerebral regional AchE activity may, therefore,contribute greatly to clinical diagnosis, therapeutic evaluation andpathological elucidation of dementing and/or age-related neurologicaldisorders.

[0003] Conventionally, AchE activity in the brain has been determinedenzymatically or histochemically using homogenate or sections ofpostmortem brain tissue. Lipophilic acetylcholine analogs labeled withradionuclide have also been used to determine AchE activity in the brainby using autoradiagraphy or emission tomography (Japanese PatentApplication laid-open No. 327497/1994). The emission tomographic methodallows non-invasive determination of AchE activity in the living brainin both human and animal subjects. This method has been found to be ofmerit for clinical diagnosis or development of therapeutic drugs fordegenerative disorders of cholinergic nerve system including Alzheimer'sdisease (Namba et. el., Brain Res., 667:278-282, 1994, Irie et. al., J.Nucl. Med., 37:649-655, 1996).

[0004] The radiolabeled compounds used in the method described in abovepublications must have following characteristics:

[0005] (1) Highly lipophilic to pass through the blood-brain barriereasily;

[0006] (2) Being specifically hydrolyzed by AchE in the brain;

[0007] (3) Being hydrolyzed to less lipophilic alcohol that is trappedin the brain; and

[0008] (4) Negligible cerebral incorporation of the hydrolyzed alcoholformed outside the brain.

[0009] The above application have shown the following radiolabeledcompounds satisfying the above requirements;N-methylpiperidinyl-3-acetate, N-methylpiperidinyl-3-propionate,N-methylpiperidinyl-4-acetate and N-methylpiperidinyl-4-propionate, eachof which has N-methyl group labeled with ¹⁴C. By using these compounds,autoradiographic determination of cerebral AchE activity ahs beenachieved in rats. Furthermore, positron emission tomography (PET) usingthe ¹¹C labeled compound has been done for non-invasive determination ofcerebral AchE activity in living subjects (Iyo el. Al., Lancet,349:1805-1809, 1997)

[0010] The conventional lipophilic acetylcholine analogs including thecompounds described above, however, allow only ¹¹C-labeling to givepractically available radiolabeled compounds used for non-invasivedetermination of cerebral AchE activity. Therefore, PET using positroncamera is the only selection allowed to be used for clinical applicationto human subjects. Because of the short half-life of ¹¹C (about 20 min),PET scanning is restricted to performing in a facility with a cyclotronfor radioisotope production. Meanwhile, single photon emission computedtomography (SPECT) using gamma camera is widely used for clinicalpractice, so the development of a radiolabeled compound applicable toSPECT has been demanded.

DISCLOSURE OF THE INVENTION

[0011] The present inventors have performed earnest studies on compoundslabeled with an appropriate gamma-ray emitting radionuclide with a viewto developing an AchE activity imaging SPECT agent fulfilling therequired characteristics, and have found novel N-alkylpiperidinederivatives labeled with radioactive iodine.

[0012] The present invention provides N-alkylpiperidine derivatives andtheir salts represented by the following general formula (1) or (2);

[0013] wherein R¹ represents a lower alkyl group which may besubstituted by a fluorine atom; R² represents a lower alkyl group; andR³ represents an alkenyl group which is substituted at its 1-position bya hydroxy group, a lower alkoxy group, a lower alkoxyalkyloxy group, alower alkoxyalkyloxyalkyloxy group, or a lower alkanoyloxy group and issubstituted at the end by radioactive iodine, or an alkenyloxymethylgroup which is substituted at an the end by radioactive iodine.

[0014] The present invention also provides a reagent for thedetermination of AchE activity including the N-alkylpiperidinederivatives and their salts.

[0015] The present invention also provides the method for thedetermination of cerebral regional AchE activity using theN-alkylpiperidine derivatives and their salts.

[0016] The present invention also provides precursors of the radioactiveN-alkylpiperidine derivatives and their salts, represented by thefollowing general formula (1P) or (2P);

[0017] wherein R¹ represents an lower alkyl group which may besubstituted by a fluorine atom; R² represents a lower alkyl group; andR^(3P) represents an alkenyl group which is substituted at its1-position by a hydroxy group, a lower alkoxy group, a loweralkoxyalkyloxy group, a lower alkoxyalkyloxyalkyloxy group, or a loweralkanoyloxy group and is substituted at the end by a non-radioactivehalogen atom, a trialkyltin group, or a trialkylsilyl group, or analkenyloxymethyl group which is substituted at the end bynon-radioactive halogen atom, a trialkyltin group, or a trialkylsilylgroup.

BRIED DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 shows the cerebral distribution of radioactivity in ratsdetermined by using dissected brain tissue after the administration ofone of the compounds produced by the present invention.

[0019]FIG. 2 shows cerebral distribution of radioactivity in ratsdetermined by quantitative autoradiography after the administration ofone of the compounds produced by the present invention.

[0020]FIG. 3 shows autoradiographic images of cerebral distribution ofradioactivity in rats after the administration of one of the compoundsproduced by the present invention.

BEST MODES FOR CARRYING OUT THE INVENTION

[0021] In the above-described formulas (1), (2), (1P), and (2P),examples of the lower alkyl groups which are represented by R¹ and whichmay be substituted by a fluorine atom include C1-C5 alkyl groups whichmay be substituted by a fluorine atom, and of these, methyl, ethyl, andfluoroethyl groups are preferred.

[0022] Examples of the lower alkyl groups represented by R² includeC1-C5 alkyl groups, and of these, methyl, ethyl, propyl and isopropylgroups are preferred.

[0023] Examples of the lower alkoxy groups at the 1-position of R³ andR^(3P) include C1-C5 alkoxy groups, and of these, a methoxy group ispreferred. Examples of the lower alkoxyalkyloxy groups at the 1-positionof R³ and R^(3P) include C₁₋₅ alkoxy-C₁₋₅ alkyloxy groups, and of these,methoxymethyloxy, ethoxymethyloxy, and ethoxyethyloxy groups arepreferred. Examples of the lower alkoxyalkyloxyalkyloxy groups at the1-position of R³ and R^(3P) include C₁₋₅ alkoxy-C₁₋₅ alkyloxy-C₁₋₅alkyloxy groups, and of these, methoxyethyloxymethyloxy andethoxyethyloxymethyloxy groups are preferred. Examples of the loweralkanoyloxy groups at the 1-position of R³ and R^(3P) include C2-C6alkanoyloxy groups, and of these, acetoxy and propionyloxy groups arepreferred.

[0024] Examples of radioactive iodine atoms substituted at the end of R³include ¹²³I and ¹³¹I, and of these, ¹²³I is preferred. Preferredexamples of non-radioactive halogen atoms substituted at the end ofR^(3P) include bromine and iodine atoms. Examples of alkenyl groups inR³ and R^(3P) include C2-C8 alkenyl groups, and of these, propenyl,butenyl, and pentenyl groups are preferred. Examples of alkenyloxymethylgroups in R³ and R^(3P) include C3-C9 alkenyloxymethyl groups, and ofthese, propenyloxymethyl and butenyloxymethyl groups are preferred, andeach of these compounds preferably has a double bond at the end. Inaddition, an alkyl group in a trialkyltin or trialkylsilyl group ispreferably a C1-C5 alkyl group.

[0025] R^(3P) is preferably a C2-C8 alkenyl group which is substitutedat the 1-position by a hydroxy group, a lower alkoxyalkyloxy group, alower alkoxyalkyloxyalkyloxy group, or a lower alkanoyloxy group, and issubstituted by a non-radioactive halogen atom, a trialkyltin group, or atrialkylsilyl group at the end. R³ is preferably a C2-C8 alkenyl groupwhich is substituted at the 1-position by a hydroxy group, a loweralkoxyalkyloxy group, a lower alkoxyalkyloxyalkyloxy group, or a loweralkanoyloxy group, and is substituted by a radioactive iodine atom atthe end.

[0026] Particularly preferred examples of the compound (1) of thepresent invention includeN-methyl-3-acetoxy-4-(1-hydroxy-3-¹²³I-2-propenyl)piperidine,N-methyl-3-acetoxy-4-(1-hydroxy-5-¹²³I-4-pentenyl)piperidine,N-methyl-4-acetoxy-3-(1-hydroxy-3-¹²³I-2-propenyl)piperidine,N-methyl-4-acetoxy-3-(1-hydroxy-5-¹²³I-4-pentenyl)piperidine,N-methyl-3-acetoxy-4-(1-methoxymethyloxy-3-¹²³I-2-propenyl)piperidine,N-methyl-3-acetoxy-4-(1-methoxymethyloxy-5-¹²³I-4-pentenyl)piperidine, N-methyl-4-acetoxy-3-(1-methoxymethyloxy-3-¹²³I-2-propenyl)piperidine,andN-methyl-4-acetoxy-3-(1-methoxymethyloxy-5-¹²³I-4-pentenyl)piperidine.

[0027] Examples of salts of the compounds (1), (2), (1P), and (2P) ofthe present invention include salts of inorganic acids such ashydrochloric acid, and salts of organic acids such as acetic acid.

[0028] Each of the compounds (1), (2), (1P), and (2P) of the presentinvention or salts thereof has two or three asymmetric carbon atoms inthe structure, and therefore has a plurality of optical isomers. Inaddition, each of the optical isomers has a plurality of geometricalisomers, in accordance with the position of the halogen atom,trialkyltin group, or trialkylsilyl group at the end of R³ or R^(3P).However, the present invention encompasses these optical isomers,geometrical isomers, and mixtures of these isomers.

[0029] The N-alkylpiperidine derivatives (1) or (2) of the presentinvention or salts thereof can be easily synthesized from theabove-described precursors (1P) or (2P), or from the salts of theprecursors. In this case, a non-radioactive halogen atom of thehalogen-containing precursor is substituted through exchange reaction bya radioactive iodine atom, or a trialkyltin group of thetrialkyltin-containing precursor or a trialkylsilyl group of thetrialkylsilyl-containing precursor is directly substituted by aradioactive iodine atom, to thereby obtain the derivatives or the salts.

[0030] The precursors shown by formulas (1P) or (2P) can be produced,for example, through the following production process (A) or (B).

[0031] wherein R¹ and R² are the same as described above; R⁴ representsan amino protective group such as a t-butoxycarbonyl group; R⁵represents a terminal alkenyl group; R^(5′) represents a terminalalkenylene group; R⁶ represents a lower alkyl group, a lower alkoxyalkylgroup, or a lower alkoxyalkyloxyalkyl group; R^(6′) represents a loweralkyl group, a lower alkoxyalkyl group, a lower alkoxyalkyloxyalkylgroup, or a lower alkanoyl group; R⁷ represents a C1-C5 alkyl group; andX¹, X², X³, X⁴, and X⁵ represent halogen atoms.

[0032] An amino protective compound of 4-piperidone (a) is reacted withmorpholine to form an enamine (b), and the thus-formed enamine isreacted with an alkynecarboxylic acid halide (c) to thereby obtain adiketone (d). The diketone (d) is reacted with a reducing agent such assodium borohydride to form a diol (e), which in turn is reacted with analkoxyalkyloxyalkyl halide, an alkoxyalkyl halide, or an alkyl halide(f) to thereby obtain a compound (g). Subsequently, the compound (g) isreacted with a carboxylic acid halide (h) to thereby obtain a compound(i). In this case, when the diol (e) is reacted with the carboxylic acidhalide, the compound (i) having a lower alkanoyl group serving as R^(6′)is obtained. An amino protective group of the compound (i) is eliminatedto form a compound (j), and the thus-formed compound (j) is reacted withan alkyl halide (k) to thereby obtain an N-alkyl substituted compound(l). In this case, N-alkylation can be performed by condensation betweenthe amino group and formaldehyde, followed by reduction. The N-alkylsubstituted compound (l) is reacted with a trialkyltin hydride or atrialkylsilane (m) to thereby obtain the trialkyltin precursor ortrialkylsilyl precursor (2Pa) of the present invention. In addition, atrialkyltin group or a trialkylsilyl group of the precursors may besubstituted by a halogen atom, and R^(6′) may be appropriatelyeliminated to thereby obtain other precursors (2Pb), (2Pc), and (2Pd).

[0033] In production process (A), for example, the compound (a) may bereacted with a trialkylsilyl halide to form atrialkylsilyloxytetrahydropyridine, which may subsequently be reactedwith a dialkoxyalkane to thereby obtain the compound (g) having analkoxy group serving as R⁶.

[0034] wherein R¹, R², R⁴, R⁷, X⁴, and X⁵ are the same as describedabove; R⁸ represents a lower alkyl group; R⁹ represents a protectivegroup of hydroxyl group, such as a lower alkoxyalkyl group; R¹⁰represents a terminal alkynyl group; R^(10′) represents a terminalalkenylene group; and X⁶ and X⁷ represent halogen atoms.

[0035] A 3-piperidone derivative (n) is reacted with a reducing agentsuch as sodium borohydride to thereby obtain a hydroxypiperidinederivative (o). The hydroxyl group of the thus-obtained derivative (o)is protected to form a compound (q), which is subsequently reduced tothereby obtain an alcohol (r). The thus-obtained alcohol is reacted withan alkyl halide (s) to form a compound (t), after which the compound (t)is reacted with a trialkyltin compound or a trialkylsilyl compound tothereby obtain a compound (u). The trialkyltin or trialkylsilyl group ofthe compound (u) is substituted by a halogen atom, and the amino groupis deprotected to thereby obtain a compound (w). Subsequently, thecompound (w) is reacted with an alkyl halide (k) to form the N-alkylsubstituted compound (l), after which the protective group of thehydroxyl group at the 3 position is eliminated, and the thus-obtainedcompound is reacted with a carboxylic acid anhydride (z), to therebyobtain the halogen-containing precursor (1Pa) of the present invention.

[0036] In addition, the alcohol (r) may be oxidized to form a4-formylpiperidine compound, and the compound may be reacted withtrialkylsilylalkynyllithium to thereby obtain a4-(α-hydroxyalkynyl)piperidine compound. The thus-obtained compound maybe subjected to trialkyltin substitution, halogenation, hydrolysis, andalkanoylation in the same way as in the above production process (A) or(B), to thereby obtain the compound of formula (1P) having a hydroxylgroup or a lower alkoxy group at the 1-position of R^(3P).

[0037] Process (A) describes the production process of the compound offormula (2P), wherein R^(3P) is an alkenyl group having a substituent atthe 1-position. By the same way as in process (A), the compound offormula 1P, wherein R^(3P) is an alkenyl group having a substituent atthe 1-position, can be produced. Meanwhile, process (B) describes theproduction process of the compound of formula (1P), having analkenyloxymethyl group serving as R^(3P). By the same way as in process(B), the compound of formula (2P), having an alkenyloxymethyl groupserving as R^(3P), can be obtained.

[0038] As is described above, the N-alkylpiperidine derivatives (1) or(2) of the present invention or salts thereof can be easily obtainedfrom the above-described precursors of formula (1P) and (2P) or saltsthereof, wherein a non-radioactive halogen atom, a trialkyltin group, ora trialkylsilyl group of the precursors or salts thereof is substitutedby a radioactive iodine atom.

[0039] Compounds (1) and (2) and the salts thereof obtained hereinabovemeet the above-described four requirements as reagents for assayingcentral AchE activity, and are radioactive compounds emitting γ-rays atan appropriate energy level for SPECT, but each compound or each isomerhas its own distinctive rate of hydrolysis. Accordingly, preferably acompound or an isomer which has high reactivity and specificity for AchEis selectively used on the occasion of application to SPECT.

[0040] Central local AchE activity can be calculated by the followingmethod: reagents for assaying central AchE activity containing compounds(1), (2), or a salt thereof are administered; after the lapse of apredetermined time, radioactive concentration in a central local site isassayed by SPECT or autoradiography by use of a central tissue slice;while blood flow rate in the central local site is assayed; and from therelationship between these assayed values and AchE activity, centrallocal AchE activity is calculated.

[0041] The blood flow rate in the central local site may be convenientlyassayed by a reference sample method making use of ¹²³I-labeledN-isopropyl-p-iodoamphetamine (IMP) (Lear et al., J. Cereb. Blood FlowMetabol. 2: 179-185, 1882; Kuhl et al., J. Nuc. Med. 23: 196-203,1982),and other known methods may also be employed.

[0042] In order to calculate central local AchE activity from thethus-assayed radioactive concentration and blood flow rate in thecentral local site, there is constructed a kinetic model incorporatingdistribution of the tracer in the central tissue depending on bloodflow, as well as the metabolic process. The manner of incorporation of aradioactive tracer (hereinafter referred to as “tracer”) into thecentral tissue differs depending on the blood flow rate, and theincorporated tracer in the central tissue is hydrolyzed competitivelywith acetylcholine by AchE into alcohols. In contrast, alcohols formedin the blood do not migrate to the central tissue. By use of adifferential equation, this is expressed by the following equations (a).$\begin{matrix}{{\frac{C_{b}}{t} = {{F( {C_{p} - {\frac{1}{\lambda}C_{b}}} )} - {\frac{V_{m}}{{K_{m}( {1 + {C_{a}/K_{ma}}} )} + C_{b}}C_{b}}}}{\frac{C_{m}}{t} = {{\frac{V_{m}}{{K_{m}( {1 + {C_{a}/K_{ma}}} )} + C_{b}}C_{b}} - {k_{e\quad 1}C_{m}}}}} & (a)\end{matrix}$

[0043] In the above formulas, C_(b) represents the concentration of anunchanged tracer in the central tissue, C_(p) represents theconcentration of an unchanged tracer in the blood, F represents theblood flow rate in the central local site, and λ represents thedistribution coefficient of brain blood in the equilibrium state of thetracer. F=λK holds true. K is the permeation velocity coefficient of atracer at the blood-brain barrier (BBB). This theoretical equation forthe blood-flow related incorporation of a tracer into the central tissueis the same as a theoretical equation for assaying the blood flow ratein the central local site by use of antipyrine iodide (Sakurada et al.,Am. J. Physiol., 234: H59-66,1978). With regard to the incorporationinto the central tissue depending on blood flow, many theoreticalequations have been proposed, and a suitable one should be useddepending on the employed tracer. Vm and Km represent the maximumhydrolysis rate and Michaelis constant of a tracer by AchE,respectively; C_(a) represents concentration of free acetylcholine inthe central tissue; K_(ms) represents the Michaelis constant ofhydrolysis of acetylcholine; C_(m) represents concentration of alcoholsin the central tissue, which alcohols are metabolites of the tracer; andk_(el) represents the disappearance velocity constant of alcohols fromthe central tissue. Acetylcholine in the central tissue is usuallyaccumulated in the synapse and discharged in the case of nerveconduction, and therefore, concentration of free acetylcholine C_(a) canbe qualified as nearly zero. Also, because the quantity of tracers usedfor the assay is trace-level, C_(b) is far less than K_(m) and can beignored. Further, alcohols disappear extremely slowly in the brain, andtherefore k_(el) can be qualified as zero. Subsequently, the abovesimultaneous differential equations (a) can be rewritten as follows.$\begin{matrix}{{\frac{C_{b}}{t} = {{F( {C_{p} - {\frac{1}{\lambda}C_{b}}} )} - {K_{m}C_{b}}}}{\frac{C_{m}}{t} = {k_{m}C_{b}}}} & (b)\end{matrix}$

[0044] In the above formulas, k_(m)(=V_(m)/K_(m)) represents theactivity of a tracer in terms of hydrolysis by AchE in the centraltissue. When a tracer is injected intravenously and rapidly, transitionof concentration of the unchanged tracer in the blood is expressed asC_(p)=ΣC_(oi)exp(−k_(ei)t), and the following equation (c) is derived.$\begin{matrix}{{C_{b} = {\sum{\frac{\lambda \quad {KC}_{oi}}{k_{ei} - ( {K + k_{m}} )}( {{\exp ( {{- ( {K + k_{m}} )}t} )} - {\exp ( {{- k_{ei}}t} )}} )}}}{C_{m} = {\sum{\frac{\lambda \quad {Kk}_{m}C_{oi}}{k_{ei} - ( {K + k_{m}} )}( {\frac{1 - {\exp ( {{- ( {K + k_{m}} )}t} )}}{K + k_{m}} - \frac{1 - {\exp ( {{- k_{ei}}t} )}}{K_{ei}}} )}}}} & (c)\end{matrix}$

[0045] If time t is sufficiently large, the exponent can be qualified aszero. In other words, concentration of an unchanged tracer in the bloodand the central tissue is qualified as zero. On this occasion, solelyalcohols, being metabolites, are present, and their concentration isexpressed by the following equation (d). $\begin{matrix}\begin{matrix}{C_{m} = {\frac{\lambda \quad {Kk}_{m}}{K + k_{m}}{\sum\frac{C_{oi}}{k_{ei}}}}} \\{= {{AUC}\frac{\lambda \quad {Kk}_{m}}{K + k_{m}}}}\end{matrix} & (d)\end{matrix}$

[0046] Accordingly, after a tracer labeled with a radioactive element isadministered intravenously, at the time point when concentration of anunchanged tracer in the blood and the central tissue becomes zero,radioactive concentration in the central tissue varies depending on AUC(area under concentration curve in the blood) of concentration of anunchanged tracer in the blood, distribution coefficient of a tracer inthe brain blood, blood flow rate in the central local site, and centrallocal AchE activity.

[0047] This is a basic equation for dynamic analysis of a tracer, but inorder to obtain k_(m), numerical values must be assayed for the AUC ofconcentration of an unchanged tracer in the blood and the blood flowrate in the central local site. However, because a tracer is rapidlydecomposed by esterase in the blood, assaying concentration of anunchanged tracer takes time. Therefore, there was examined an assayingmethod in which corpus striatum having high AchE activity is employed asan internal standard. Corpus striatum is assigned an affix “s,” and aregion of interest in the brain is assigned an affix “o.” A ratio ofradioactive concentration in the central tissue is expressed by thefollowing equation (e): $\begin{matrix}{\frac{C_{o}}{C_{s}} = {\frac{\lambda_{o}K_{o}k_{mo}}{K_{o} + k_{mo}}\frac{K_{s} + k_{ms}}{\lambda_{s}K_{s}k_{ms}}}} & (e)\end{matrix}$

[0048] In this case, AUCs cancel each other out. Herein, C_(o)/C_(s) isreplaced by Z to thereby obtain the following equation (f):$\begin{matrix}{\frac{1}{Z} = {\frac{K_{s}k_{ms}}{K_{s} + k_{ms}}\frac{\lambda_{s}}{\lambda_{o}}( {\frac{1}{K_{o}} + \frac{1}{k_{mo}}} )}} & (f)\end{matrix}$

[0049] In addition, the equation may be rewritten to thereby obtain thefollowing equation (g). $\begin{matrix}{\frac{1}{Z} = {{\frac{k_{ms}}{K_{s} + k_{ms}}\frac{\lambda_{s}K_{s}}{\lambda_{o}K_{o}}} + {\frac{K_{s}k_{ms}}{K_{s} + k_{ms}}\frac{\lambda_{s}}{\lambda_{o}}\frac{1}{k_{mo}}}}} & (g)\end{matrix}$

[0050] wherein λ_(o)K_(o)/λ_(s)K_(s) refers to the ratio of the bloodflow rate in a region of interest to that in the corpus striatum, andk_(mo) refers to hydrolysis activity of a tracer in the region ofinterest. Herein, λ_(o)K_(o)/λ_(s)K_(s) and k_(mo), are replaced by Fand Y, respectively, to thereby obtain the following equation (h):$\begin{matrix}{\frac{1}{Z} = {{\frac{k_{ms}}{K_{s} + k_{ms}}\frac{1}{F}} + {\frac{K_{s}k_{ms}}{K_{s} + k_{ms}}\frac{\lambda_{s}}{\lambda_{o}}\frac{1}{Y}}}} & (h)\end{matrix}$

[0051] and the equation is solved for 1/Y, to thereby obtain thefollowing equation (i): $\begin{matrix}\begin{matrix}{\frac{1}{Y} = {{\frac{K_{s} + k_{ms}}{K_{s}k_{ms}}\frac{\lambda_{o}}{\lambda_{s}}\frac{1}{Z}} - {\frac{1}{K_{s}}\frac{\lambda_{o}}{\lambda_{s}}\frac{1}{F}}}} \\{= {\frac{\lambda_{o}}{\lambda_{s}}\lbrack {{( {\frac{1}{k_{ms}} + \frac{1}{K_{s}}} )\frac{1}{Z}} - {\frac{1}{K_{s}}\frac{1}{F}}} \rbrack}}\end{matrix} & (i)\end{matrix}$

[0052] In this case, Y refers to the hydrolysis activity of a tracer.Therefore, metabolic activity y of acetylcholine by AchE is obtainedfrom the following equation (j), wherein the ratio of metabolic activityof the tracer kmo to that of acetylcholine k_(ma); i.e., k_(mo)/k_(ma),is replaced by φ: $\begin{matrix}\begin{matrix}{\frac{1}{y} = {\varphi {\frac{\lambda_{o}}{\lambda_{s}}\lbrack {{( {\frac{1}{k_{ms}} + \frac{1}{K_{s}}} )\frac{1}{Z}} - {\frac{1}{K_{s}}\frac{1}{F}}} \rbrack}}} \\{= {{A\frac{1}{Z}} - {B\frac{1}{F}}}}\end{matrix} & (j)\end{matrix}$

[0053] In the equation, the two parametersA=φ(1/k_(ma)+1/K_(s))(λ_(o)/λ_(s)) and B=φ(1/K_(s))(λ_(o)/λ_(s)) areunknown quantities. However, in animals, AchE activity y and the corpusstriatum ratio of radioactive concentration in the central tissue Z canbe practically measured by use of a tissue slice of brain tissue, whichis obtained by punch-out.

[0054] When [¹²³I]IMP, serving as a tracer, is used for measurement ofthe blood flow rate in the central local site, the amount is representedby C_(b)/AUC (C_(b): radioactive concentration of the central localsite, AUC: concentration of unchanged IMP in the blood), and the corpusstriatum ratio of blood flow in the central local site F becomesequivalent to the corpus striatum ratio of ¹²³I concentration. Thus, theblood flow rate in the central local site can be practically measured byuse of a tissue slice of the brain, which is obtained by punch-out.Therefore, in regions of the brain and corpus striatum, if the ratio ofpartition coefficient of tracer λ_(o)/λ_(s) is almost the same in theregions, the unknown parameters A and B can be determined throughapplication of the linear least square method to practically-measured y,Z, and F. In addition, AUC may be practically measured by use ofconcentration of unchanged IMP in the blood, the absolute value of bloodflow in the central local site may be practically measured, and km maybe practically measured by use of brain tissue obtained by punch-out. Byuse of these practically-measured values and the above equation (d), allparameters, including λ and K, may be determined.

[0055] In the case of animals, since AchE activity can be practicallymeasured in vitro, a variety of techniques are applicable todetermination of parameters, and studies making use of pathologicanimals are also available. Meanwhile, in the case of human subjects,mean values of parameters of a healthy human are obtained and used. Inorder to obtain the mean value for a healthy human, k_(m) and φ aremeasured by use of the autopsied brain of a person who has perished inan accident. In addition, a tracer is administered to a healthy humanand C_(m) is measured through PET, to thereby obtain AUC. By use ofthese values and the blood flow rate in the central local site of thehealthy human, λ and K are obtained through equation (d). Thethus-obtained values can be used for determination of the mean values ofparameters A and B of the healthy human.

EXAMPLES

[0056] The present invention will next be described in more detail byway of examples, which should not be construed as limiting the inventionthereto.

Example 1

[0057] (1) Water (250 ml) was added to 4-piperidone monohydratehydrochloride salt (75 g) for dissolution, and 1 N aqueous solution ofsodium hydroxide (1000 ml) was added thereto. To the solution,ditertiarybutylcarbonate (120 g) was added dropwise with stirring undercooling on ice, and the mixture was vigorously stirred for 6 hours atroom temperature. The reaction mixture was subjected to extraction withethyl acetate, and the extract was evaporated to dryness under reducedpressure to thereby yield a pale yellow solid. By recrystallization ofthe solid from hexane, N-t-butyloxycarbonyl-4-piperidone (3) (38.9 g)was obtained in the form of white needle-shaped crystals.

[0058] mp.: 74.4-75.2° C.

[0059] (2) To compound No. 3 (30.0 g) obtained in (1), toluene (150 ml)and morpholine (19 ml) were added, and the mixture was refluxed withheat for 20 hours in an atmosphere of nitrogen gas in a Dean-Starkreflux apparatus. After the mixture was allowed to cool, it wasevaporated to dryness to thereby yield enamine (4) in the form of a paleyellow semi-solid.

[0060] (3) Absolute dioxane (150 ml) was added to the entirety ofenamine (4) obtained in (2) for dissolution, and 4-pentinoyl chloride(6.0 g) which had been synthesized from 4-pentinic acid and thionylchloride was added dropwise thereto with stirring. The mixture wasstirred with reflux with heat for 16 hours under an atmosphere ofnitrogen gas. After the mixture was allowed to cool to room temperature,the resultant precipitate was separated by filtration and the filtratewas concentrated under reduced pressure to thereby obtain areddish-brown oil. The oil was purified by silica gel chromatography(hexane:ethyl acetate=4:1) and recrystallized from hexane to therebyyield N-t-butyloxycarbonyl-3-(1-oxo-4-pentinyl)-4-piperidone (5) (6.03g) in the form of colorless needle-shaped crystals.

[0061] mp.: 76.8-77.6° C.

[0062] High resolution mass spectrum (electron impact mode, [M−C₄H₉]⁺):found: 222.0744 calculated: 222.0765.

[0063] (4) Diketone (5) (5.58 g) obtained in (3) was dissolved in a 1:1mixture (80 ml) of ethyl acetate and ethanol, and powdery sodium boronhydride (1000 mg) was added thereto portionwise with stirring at roomtemperature, followed by stirring for 1 hour. The reaction mixture wasdiluted with water and subjected to extraction with ethyl acetate. Theextract was washed with water, dried over anhydrous sodium sulfate, andconcentrated under reduced pressure to thereby yieldN-t-butyloxycarbonyl-3-(1-hydroxy-4-pentinyl)-4-piperidinol (6) (4.82 g)in the form of a colorless oil.

[0064] High resolution mass spectrum (secondary ion mode, [M+H]⁺):found: 284.1855. calculated: 284.1860

[0065] The diol (6) has 3 asymmetrical carbons and is a mixture of 8kinds of optical isomers. These optical isomers were separated into 3diastereomer fractions by silica gel chromatography (hexane:ethylacetate=1:1). According to the order of elution, the eluate wascollected separately as fractions (6a), (6b), and (6c), and eachfraction was subjected to the following synthesis to thereby yieldrespective compounds which have different reactivities for AchE.

[0066] (5) To fraction (6a) (1.44 g) of diol obtained in (4), ethylacetate (30 ml) and diisopropylethylamine (8.9 ml) were added, andfurther, chloromethylmethyl ether (2.0 ml) was added thereto dropwisewith stirring, followed by stirring for 5 hours at room temperature. Tothe mixture, water was added under cooling on ice, and after 1 Nhydrochloric acid (30 ml) was added dropwise thereto to thereby make themixture acidic, the mixture was subjected to extraction with ethylacetate. The extract was washed with water, dried over anhydrous sodiumsulfate, and concentrated under reduced pressure to thereby yield acolorless oil. The oil was purified by silica gel chromatography(hexane:ethyl acetate=1:1) to thereby obtainN-t-butyloxycarbonyl-3-(1-methoxymethyloxy-4-pentinyl)-4-piperidinol(7). Further, unreacted diol (6a) was recovered from the column andagain subjected to a methoxymethylation reaction and to columnchromatography to thereby yield compound No. 7. Total yield of compoundNo. 7 was 0.60 g.

[0067] The obtained compound No. 7 is a mixture of two different opticalisomers, and each enantiomer can be separated into a first isomer (7a)which elutes first and a second isomer (7b) which elutes later, by meansof elution with an eluent (hexane:2-propanol=9:1) by use of Chiral HPLC(CHIRALCEL OJ column; DAICEL CHEMICAL INDUSTRIES, LTD.). By subjectingeach isomer to the following synthetic method, compounds havingdifferent reactivity for AchE can be obtained.

[0068] Isomer (7a)

[0069] High resolution mass spectrum (secondary ion mode, [M+H]⁺):found: 328.2105 calculated: 328.2122.

[0070] Angle of rotation: 90.0° [α]_(D)(c=0.25%, EtOAc)

[0071] Isomer (7b)

[0072] High resolution mass spectrum (secondary ion mode, [M+H]⁺):found: 328.2114 calculated: 328.2122.

[0073] Angle of rotation: 91.6° [α]_(D)(c=0.25%, EtOAc)

[0074] (6) Isomer (7b) obtained in (5) (216 mg) was dissolved in a 1:1mixture of hexane and methylene chloride (40 ml), andN,N-dimethylaminopyridine (806 mg) was added thereto. Acetyl chloride(235 μl) was added dropwise to the mixture with stirring, and theresultant mixture was stirred for 5 hours at room temperature. After themixture was cooled on ice, water was added thereto, and further, 1 Nhydrochloric acid (30 ml) was added dropwise to thereby make the mixtureacidic. An organic phase was separated, washed with water and then withaqueous saturated brine, and dried over anhydrous sodium sulfate,followed by concentration under reduced pressure to thereby yield acolorless oil. The oil was purified by silica gel chromatography(hexane:ethyl acetate=3:1) to thereby obtainN-t-butyloxycarbonyl-3-(1-methoxymethyloxy-4-pentinyl)-4-acetoxypiperidine(8) (250 mg).

[0075] High resolution mass spectrum (electron impact mode, M⁺): found:369.2160 calculated: 369.2150.

[0076] (7) Compound No. 8 (125 mg) obtained in (6) was dissolved in a1:1 mixture of hexane and methylene chloride (20 ml), andtrifluoroacetic acid (1.0 ml) was added dropwise to the solution withstirring, and further, the resultant mixture was stirred for 16 hours atroom temperature. The mixture was concentrated under reduced pressure,and methylene chloride (20 ml) was added to the resultant residue. Aftera few drops of a concentrated ammonia water were added thereto withstirring under cooling on ice, the organic phase was separated and driedover anhydrous sodium sulfate, followed by concentration under reducedpressure to thereby yield3-(1-methoxymethyloxy-4-pentinyl)-4-acetoxypiperidine (9) (90 mg) in theform of a colorless oil.

[0077] (8) Compound No. 9 (90 mg) obtained in (7) was dissolved inacetone (20 ml), methyl iodide (25 mg) was added dropwise to thesolution under cooling at −80° C. with stirring, and the resultantmixture was stirred for 2 hours at room temperature. The mixture wasconcentrated under reduced pressure, and chloroform (15 ml) was added tothe resultant residue. After a drop of aqueous concentrated solution ofammonia was added thereto under cooling on ice with stirring, theresultant mixture was dried over anhydrous sodium sulfate. The driedmatter was purified by silica gel chromatography(chloroform:methanol=15:1) to thereby obtainN-methyl-3-(1-methoxymethyloxy-4-pentinyl)-4-acetoxypiperidine (10) (17mg).

[0078] High resolution mass spectrum (electron impact mode, M⁺): found:283.1818 calculated: 283.1782.

[0079] (9) Compound No. 10 (17 mg) obtained in (8) and2,2′-azobisisobutyronitrile (5 mg) were dissolved in absolute toluene(10 ml), and hydrogenated tributyltin (100 μl) was added dropwise to thesolution in an atmosphere of nitrogen gas, after which the resultantmixture was stirred for 1 hour at 85° C. The mixture was distilled offunder reduced pressure, and the resultant residue was purified by silicagel chromatography (chloroform:methanol 15:1) to thereby yieldN-methyl-3-(1-methoxymethyloxy-5-tributylstannyl-4-pentinyl)-4-acetoxypiperidine(11) (30 mg).

[0080] (10) Compound No. 11 (30 mg) obtained in (9) was dissolved inmethylene chloride (5 ml), and a solution of N-iodosuccinimide dissolvedin methylene chloride was gradually added dropwise to the mixture withstirring under cooling on ice. At a point of time when compound No. 11disappeared, the solvent was distilled off under reduced pressure andthe resultant residue was purified by silica gel chromatography(chloroform:methanol=15:1) to thereby yieldN-methyl-3-(1-methoxymethyloxy-5-iodo-4-pentinyl)-4-acetoxypiperidine(12) (16 mg).

[0081] High resolution mass spectrum (electron impact mode, M⁺): found:411.0908 calculated: 411.0905.

[0082] The obtained compound No. 12 is a mixture of two differentgeometrical isomers, and the isomers can be separated into a firstisomer (12a) which elutes first and a second isomer (12b) which eluteslater, by means of elution with an eluent(methanol:water:triethylamine=70:30:0.1) by use of HPLC (ODS C18column). Each isomer has different a reactivity for AchE.

[0083] (11) Compound No. 12 (16 mg) obtained in (10) was dissolved inmethylene chloride (1 ml), and after trifluoroacetic acid (3 ml) wasadded thereto, the mixture was allowed to stand at room temperature for24 hours. The solvent was distilled off under reduced pressure and theresultant residue was purified by silica gel chromatography(chloroform:methanol=8:1) to thereby yieldN-methyl-3-(1-hydroxy-5-iodo-4-pentinyl)-4-acetoxypiperidine (13) (13mg).

[0084] High resolution mass spectrum (electron impact mode, M⁺): found:367.0600 calculated: 367.0642.

[0085] The obtained compound No. 13 is a mixture of two differentgeometrical isomers, and the isomers can be separated into a firstisomer (13a) which elutes first and a second isomer (13b) which eluteslater, by means of elution with an eluent(methanol:water:triethylamine=60:40:0.1) by use of HPLC (ODS C18column). Each isomer has a different reactivity for AchE.

Example 2

[0086] (1) N-benzyl-4-ethoxycarbonyl-3-piperidone hydrochloride salt(15.0 g) was dissolved in a 1:1 mixture (300 ml) of ethanol and water,and 10% palladium-on-carbon (1 g) was added to the solution, followed bystirring for 12 hours in an atmosphere of hydrogen gas. The catalyst wasremoved by filtration and the filtrate was concentrated under reducedpressure. To the resultant residue, water (80 ml), potassium carbonate(21 g), and dioxane (100 ml) were added, and di-t-butylcarbonate (12 g)was gradually added dropwise thereto with stirring under cooling on ice,followed by stirring vigorously for 1.5 hours. The resultant mixture wasdiluted with water and subjected to extraction with ethyl acetate. Afterthe extract was dried over anhydrous sodium sulfate, the solvent wasdistilled off under reduced pressure to thereby yieldN-t-butyloxycarbonyl-4-ethoxycarbonyl-3-piperidone (14) (14.1 g).

[0087] (2) Compound No. 14 (10.8 g) obtained in (1) was dissolved inmethanol (50 ml), and sodium boron hydride was added thereto withstirring under cooling on ice until the raw material disappeared. Thesolvent was distilled off under reduced pressure and water was added tothe resultant residue, which was then subjected to extraction with ethylacetate. The extract was dried over anhydrous sodium sulfate and thesolvent was distilled off under reduced pressure to thereby yield anoily matter. The oil was purified by silica gel chromatography(hexane:ethyl acetate=2:1) to thereby yieldN-t-butyloxycarbonyl-3-hydroxy-4-ethoxycarbonylpiperidine (15) (6.95 g).

[0088] FAB-MS(Glycerol) [M+H]⁺ 274

[0089] (3) Compound No. 15 (5.46 g) obtained in (2) was dissolved inethyl acetate (50 ml), and diisopropylethylamine (10.4 ml) were addeddropwise thereto with stirring and further, chloromethyl methyl ether(2.3 ml) was added dropwise thereto, followed by stirring for 48 hoursat room temperature. After dilution of the mixture with water, 1 Nhydrochloric acid was added thereto under cooling on ice to thereby makethe mixture acidic, and the ethyl acetate layer was collected. After theethyl acetate layer was dried over anhydrous sodium sulfate, the solventwas distilled off under reduced pressure. The resultant residue waspurified by silica gel chromatography (hexane:ethyl acetate=3:1) tothereby yieldN-t-butyloxycarbonyl-3-methoxymethyloxy-4-ethoxycarbonylpiperidine (16)(2.56 g) in the form of a colorless oil.

[0090] EI-MS M⁺ 317

[0091]¹H-NMR(CDCl₃) δ: 1.27(3H, t), 1.46(9H, s), 1.61-1.72(2H, m),1.89-1.93(1H, m), 2.44-2.52(1H, m), 2.69-2.81(2H, m), 3.35(3H, s),3.78(1H, m), 3.97(1H, t), 4.17(2H, q), 4.67(2H, s)

[0092] The obtained compound No. 16 has 2 asymmetric carbons and is amixture of four different optical isomers. These optical isomers areseparated into two diastereomer fractions by silica gel chromatography(hexane:ethyl acetate=5:1). According to the order of elution, theeluate was collected separately as fractions (16a) and (16b), and eachfraction was subjected to the following synthesis to thereby yieldrespective compounds which have different reactivities for AchE.

[0093] (4) Lithium boron hydride (200 mg) was added to absolutetetrahydrofuran (30 ml), and compound No. 16a (2.23 g) obtained in (3)was added dropwise thereto, followed by reflux with heat for 4 hours.The solvent was distilled off under reduced pressure and water was addedto the resultant residue, after which the mixture was subjected toextraction with ethyl acetate and the extract was dried over anhydroussodium sulfate. The dried matter was purified by silica gelchromatography (hexane:ethyl acetate=1:1) to thereby yieldN-t-butyloxycarbonyl-3-methoxymethyloxy-4-hydroxymethylpiperidine (17)(1.34 g) in the form of a colorless oil.

[0094] FAB-MS(Glycerol) [M+H]⁺ 276

[0095]¹H-NMR(CDCl₃) δ: 1.45(9H, s), 1.58-1.82(6H, m), 2.73(2H, bs),3.42(3H, s), 3.59-3.70(2H, m), 3.89(1H, s), 4.59(1H, d), 4.79(1H, d)

[0096] (5) Tetrahydrofuran (10 ml) was added to a 60% oil suspension ofsodium hydroxide (430 mg), and compound No. 17 (987 mg) obtained in (4),which had been dissolved in tetrahydrofuran (10 ml), was added dropwisethereto, followed by stirring for 30 minutes at room temperature.Further, propargyl bromide (390 μl) was added dropwise thereto and themixture was stirred for 16 hours at room temperature. Under cooling onice, water was added, and then, 1 N hydrochloric acid was added tothereby make the mixture acidic. The mixture was subjected to extractionwith ethyl acetate, and the extract was dried over anhydrous sodiumsulfate. The dry matter was purified by silica gel chromatography(hexane:ethyl acetate=3:1) to thereby yieldN-t-butyloxycarbonyl-3-methoxymethyloxy-4-propargyloxymethylpiperidine(18) (829 mg) in the form of a colorless oil.

[0097] EI-MS M⁺ 313

[0098]¹H-NMR(CDCl₃) δ: 1.45(9H, s), 1.85-1.94(1H, bs), 2.42(2H, t),2.71(2H, bs), 3.40(3H, s), 3.58(1H, t), 3.84(1H, s), 4.14(2H, d),4.60(1H, d), 4.77(1H, d)

[0099] The obtained compound No. 18 is a mixture of two differentgeometrical isomers, and each enantiomer can be separated into a firstisomer (18a) which elutes first and a second isomer (18b) which eluteslater, by means of elution with an eluent (hexane:2-propanol=100:1) byuse of Chiral HPLC (CHIRALCEL 0J column). By subjecting each isomer tothe following synthesis, compounds having different reactivities forAchE can be obtained.

[0100] (6) Compound No. 18b (57 mg) obtained in (5) was dissolved inabsolute toluene (5 ml) and under N₂ gas, 2,2′-azobisisobutyronitrile (5mg) and subsequently, hydrogenated tributyltin (100 μl) was added,followed by stirring for 2 hours at 85-90° C. The mixture was purifiedby silica gel chromatography (hexane:ethyl acetate=5:1) to thereby yieldN-t-butyloxycarbonyl-3-methoxymethyloxy-4-(3-tributylstannyl-2-propenyloxymethyl)piperidine(19) (57 mg) in the form of a colorless oil.

[0101] (7) Compound No. 19 (57 mg) obtained in (6) was dissolved inmethylene chloride (5 ml), and N-iodosuccinimide was added thereto withstirring under cooling on ice until the raw material had disappeared.The product was purified by silica gel chromatography (hexane:ethylacetate=3 1) to thereby yieldN-t-butyloxycarbonyl-3-methoxymethyloxy-4-(3-iodo-2-propenyloxymethyl)piperidine(20) (35 mg) in the form of a colorless oil.

[0102] EI-MS M⁺ 441

[0103] (8) Compound No. 20 (35 mg) obtained in (7) was dissolved inmethylene chloride (5 ml), and trifluoroacetic acid (50 μl) was added tothe solution with stirring, which was allowed to stand for 16 hours atroom temperature. The mixture was concentrated under reduced pressure,and methylene chloride (5 ml) was added to the resultant residue. Aftera drop of an aqueous concentrated solution of ammonia was added theretounder cooling on ice with stirring, the resultant mixture was dried overanhydrous sodium sulfate and concentrated. To the resultant residue,acetone (5 ml) was added, and further, methyl iodide (5 mg) was addedthereto under cooling on ice with stirring, followed by stirring for 2hours at room temperature. The product was purified by silica gelchromatography (chloroform:methanol=15:1) to thereby yieldN-methyl-3-methoxymethyloxy-4-(3-iodo-2-propenyloxymethyl)piperidine(21) (5 mg) in the form of a colorless oil.

[0104] (9) To compound No. 21 (5 mg) obtained in (8), methylene chloride(0.5 ml) and trifluoroacetic acid (1 ml) were added, and the resultantmixture was allowed to stand for 16 hours at room temperature. Themixture was concentrated under reduced pressure, and to the resultantresidue, pyridine (2 ml) and acetic anhydride (1 ml) were added, afterwhich the resultant mixture was allowed to stand for 16 hours at roomtemperature. The product was purified by silica gel chromatography(chloroform:methanol=15:1) to thereby yieldN-methyl-3-acetoxy-4-(3-iodo-2-propenyloxymethyl)piperidine (22) (5 mg)in the form of a colorless oil.

[0105] EI-MS M⁺ 353

[0106]¹H-NMR(CDCl₃) δ: 1.53-1.62(1H, m), 1.70-1.79(1H, bs),1.92-2.10(3H, bs), 2.06(3H, s), 2.35(3H, s), 2.84-2.86(1H, m),3.03-3.07(1H, m), 3.28-3.32(1H, m), 3.44-3.48(1H, m), 3.85-3.87(2H, m),4.81-4.86(1H, m), 6.36(1H, d), 6.58(1H, m)

Example 3

[0107] (1) Compound No. 6a (2.83 g) obtained in Example 1 (4) andN,N-diisopropylethylamine (8.7 ml) were dissolved in methylene chloride(50 ml), and 2-methoxyethoxymethyl chloride (2.85 ml) was added dropwisethereto, followed by stirring for 12 hours at room temperature. Aftercompletion of the reaction, water was added thereto under cooling onice, and 1 N hydrochloric acid was added dropwise thereto to therebymake the mixture acidic. Subsequently, the organic phase was removed andthe water phase was subjected to extraction with methylene chloride. Theextract was combined with the organic phase and washed with water andaqueous saturated brine. After the mixture was dried over anhydroussodium sulfate, the solvent was distilled off under reduced pressure.The resultant residue was purified by silica gel chromatography(methylene chloride:methanol=100:1) to thereby yield1-t-butoxycarbonyl-3-[1-(2-methoxyethoxy)-methoxy-4-pentinyl]-4-piperidinol(23) (2.12 g) in the form of a colorless oil.

[0108]¹H-NMR(CDCl₃) δ: 1.46(9H, s), 1.53-1.62(2H, m), 1.74-1.79(1H, m),1.94(1H, s), 2.34-2.37(2H, m), 3.09-3.20(2H, m), 3.40(3H, s),3.56-3.58(2H, m), 3.69-3.79(2H, m), 3.92-3.98(2H, bs), 4.11(1H, d),4.77(1H, d), 4.87(1H, d)

[0109] (2) Compound No. 23 (2.02 g) obtained in (1) and4-dimethylaminopyridine (2.93 g) were dissolved in methylene chloride(50 ml), and acetyl chloride (0.85 ml) was added dropwise thereto undercooling on ice, followed by stirring for 2 hours at room temperature.After completion of the reaction, water was added thereto under coolingon ice, and 1 N hydrochloric acid was added dropwise thereto to therebyneutralize the mixture. Subsequently, the organic phase was removed, andthe water phase was subjected to extraction with methylene chloride. Theextract was combined with the organic phase and washed with water andaqueous saturated brine. After the mixture was dried over anhydroussodium sulfate, the solvent was distilled off under reduced pressure.The resultant residue was purified by silica gel chromatography(n-hexane:ethyl acetate=3:1) to thereby yieldN-t-butyloxycarbonyl-3-[1-(2-methoxyethoxy)-methoxy-4-pentinyl]-4-acetoxypiperidine(24) (1.87 g) in the form of a colorless oil.

[0110] (3) Compound No. 24 (300 mg) obtained in (2) was dissolved inmethylene chloride (20 ml), and trifluoroacetic acid (1.67 ml) inmethylene chloride (5 ml) was added dropwise to the solution, followedby stirring for 30 minutes at room temperature. The reaction mixture wasconcentrated, and the resultant residue was dissolved in chloroform (10ml). After the solution was washed with aqueous saturated sodiumhydrogencarbonate and the organic phase was dried over anhydrous sodiumsulfate, the solvent was distilled off under reduced pressure. Theobtained residue in the form of a pale yellow oil was suspended in amixed solvent of acetonitrile (5 ml), water (3 ml), and methanol (5 ml).To the suspension, a 37 wt. % solution (330 μl) of formaldehyde wasadded, and further, sodium cyanoborohydride (150 mg) was added theretounder cooling on ice, followed by stirring for 15 hours at roomtemperature. The reaction mixture was concentrated, and water (10 ml)was added to the resultant residue. Acetic acid was added dropwisethereto to thereby adjust the pH of the mixture to 3, and then themixture was subjected to extraction with chloroform.

[0111] The organic phase was washed with aqueous saturated sodiumhydrogencarbonate and dried over anhydrous sodium sulfate, and then thesolvent was distilled off under reduced pressure. The obtained residuewas purified by silica gel chromatography (methylenechloride:methanol=15:1) to thereby yieldN-methyl-3-[1-(2-methoxyethoxy)-methoxy-4-pentinyl]-4-acetoxypiperidine(25) (130 mg) in the form of a colorless oil.

[0112] (4) Compound No. 25 (120 mg) obtained in (3) and hydrogenatedtri-n-butyl tin (148 μl) were dissolved in absolute toluene (5 ml), andtetrakis(triphenylphosphine) palladium (21 mg) was added to the solutionat 0° C. under argon, followed by stirring for 1 hour at roomtemperature. The mixture was concentrated, and the resultant residue wassubjected to silica gel chromatography (methylenechloride:methanol=20:1) to thereby obtain a fraction of interest. Thefraction was purified by NH silica gel chromatography (n-hexane:ethylacetate=3:1) to thereby yieldN-methyl-3-[1-(2-methoxyethoxy)methoxy-5-tributylstannyl-4-pentinyl]-4-acetoxypiperidine(26) (160 mg) in the form of a pale yellow oil.

[0113] The obtained compound No. 26 is a mixture of two differentgeometrical isomers, and the isomers can be separated into a firstisomer (26a) which elutes first and the other isomer (26b) which eluteslater, by means of elution with an eluent(methanol:water:triethylamine=90:10:0.2) by use of HPLC (ODS C18column). Each isomer has a different reactivity for AchE.

[0114] (5) Compound No. 26b (43 mg) obtained in (4) was dissolved inmethylene chloride (3 ml), and N-iodosuccinimide (25 mg) dissolved inmethylene chloride (3 ml) was gradually added dropwise to the mixture at0° C., followed by stirring for 5 minutes at the same temperature. Thesolvent was distilled off under reduced pressure, and the resultantresidue was purified by silica gel chromatography (methylenechloride:methanol=10:1) to thereby yieldN-methyl-3-[1-(2-methoxyethoxy)methoxy-5-trans-iodo-4-pentinyl]-4-acetoxypiperidine(27) (19 mg) in the form of a colorless oil.

[0115]¹H-NMR(CDCl₃) δ: 1.59-1.68(2H, m), 1.81-1.97(2H, m), 2.06(3H, s),2.08-2.23(5H, m), 2.28(3H, s) 2.52-2.60(2H, m), 3.40(3H, s), 3.54(2H,t), 3.66-3.76(2H, m), 3.86(1H, s), 4.67(1H, d), 4.80(1H, d),5.02-5.08(1H, m), 6.02(1H, d), 6.47-6.54(1H, m)

Example 4

[0116] (1) Compound No. 6a (2.27 g) and 4-dimethylaminopiperidine (5.86g) were dissolved in methylene chloride (100 ml), and acetyl chloride(1.71 ml) was added dropwise thereto under cooling on ice, followed bystirring for 2 hours at room temperature. After completion of thereaction, water was added thereto under cooling on ice, and 1 Nhydrochloric acid was added dropwise thereto for neutralization of themixture. Subsequently, the organic phase was removed and the water phasewas subjected to extraction with methylene chloride. The extract wascombined with the organic phase, and the mixture was washed with waterand aqueous saturated brine. After the mixture was dried over anhydroussodium sulfate, the solvent was distilled off under reduced pressure.The resultant residue was purified by silica gel chromatography(n-hexane:ethyl acetate=3:1) to thereby yieldN-t-butyloxycarbonyl-3-(1-acetoxy-4-pentinyl)-4-acetoxypiperidine (28)(2.05 g) in the form of a colorless oil.

[0117]¹H-NMR(CDCl₃) δ: 1.46(9H, s), 1.62-1.78(2H, m), 1.84-1.95(4H, m),2.09(3H, s), 2.11(3H, s), 2.16-2.22(2H, m), 2.92(2H, bs), 3.90(2H, bs),5.01(1H, bs), 5.20(1H, s)

[0118] (2) Compound No. 28 (1.8 g) obtained in (1) was dissolved inchloroform (100 ml), and trifluoroacetic acid (15.1 ml) was addeddropwise to the solution under cooling on ice, followed by stirring for30 minutes at room temperature. The reaction mixture was concentrated,and the resultant residue was again dissolved in chloroform (100 ml).After the solution was washed with aqueous saturated sodiumhydrogencarbonate and the organic phase was dried over anhydrous sodiumsulfate, the solvent was distilled off under reduced pressure. Theobtained residue in the form of a pale yellow oil was suspended in amixed solvent of acetonitrile (20 ml), water (10 ml), and methanol (20ml). To the suspension, a 37 wt. % solution (2.25 ml) of formaldehydewas added, and further, sodium cyanoborohydride (1.0 g) was addedthereto under cooling on ice, followed by stirring for 16 hours at roomtemperature. The reaction mixture was concentrated, and water (30 ml)was added to the resultant residue. Acetic acid was added dropwisethereto to thereby adjust the pH of the mixture to 3, and then themixture was subjected to extraction with chloroform. After the organicphase was dried over anhydrous sodium sulfate, the solvent was distilledoff under reduced pressure. The obtained residue was purified by silicagel chromatography (methylene chloride:methanol=20:1) to thereby yieldN-methyl-3-(1-acetoxy-4-pentinyl)-4-acetoxypiperidine (29) (570 mg) inthe form of a white amorphous powder.

[0119] (3) To a solution of compound No. 29 (282 mg) obtained in (2) andhydrogenated tri-n-butyltin (404 μl) in absolute toluene (10 ml),tetrakis(triphenylphosphine) palladium (58 mg) was added at 0° C. underargon gas and the resultant mixture was stirred for 1 hour at roomtemperature. The reaction mixture was concentrated, and the resultantresidue was subjected to silica gel chromatography (methylenechloride:methanol=20:1) to thereby obtain a fraction of interest. Thefraction was purified by NH silica gel chromatography (n-hexane:ethylacetate=3:1) to thereby yieldN-methyl-3-(1-acetoxy-5-tributylstannyl-4-pentinyl)-4-acetoxypiperidine(30) (460 mg) in the form of a pale yellow oil.

[0120] The obtained compound No. 30 is a mixture of two differentgeometrical isomers, and the isomers can be separated into a firstisomer (30a) which elutes first and a second isomer (30b) which eluteslater, by means of elution with an eluent(methanol:water:triethylamine=90:10:0.2) by HPLC (ODS C18 column). Eachisomer has a different reactivity for AchE.

[0121] (4) Compound No. 30b (115 mg) obtained in (3) was dissolved inmethylene chloride (3 ml), and N-iodosuccinimide (54 mg) dissolved inmethylene chloride (5 ml) was gradually added dropwise to the mixture at0° C., followed by stirring for 5 minutes at the same temperature. Thesolvent was distilled off under reduced pressure, and the resultantresidue was purified by silica gel chromatography (methylenechloride:methanol=10:1) to thereby yieldN-methyl-3-(1-acetoxy-5-trans-iodo-4-pentinyl)-4-acetoxypiperidine (31)(62 mg) in the form of pale yellow needle-shaped crystals.

[0122]¹H-NMR(CDCl₃) δ: 1.53-1.62(1H, m), 1.68-1.80(2H, m), 1.86-2.15(6H,m), 2.07(3H, s), 2.10(3H, s), 2.30(3H, s), 2.59-2.63(2H, m),4.85-4.90(1H, m), 5.06(1H, d), 6.02(1H, d), 6.42-6.49(1H, m)

Example 5

[0123] (1) To a solution of diisopropylamine (15.2 ml) in absolutetetrahydrofuran (200 ml), a 1.6 M solution of n-butyl lithium inn-hexane (60 ml) was added dropwise at -78° C. under argon gas. Thetemperature was raised to 0° C. and the mixture was stirred for 30minutes. Again the temperature was lowered to −78° C., andN-t-butyloxycarbonyl-4-piperidone (3) (17.9 g) dissolved in absolutetetrahydrofuran (30 ml) was added dropwise to the mixture, followed bystirring for 1 hour at the same temperature. Chlorotrimethylsilane (17.2ml) was added thereto, and the resultant mixture was stirred for 1 hourat room temperature. The reaction mixture was concentrated and water wasadded to the resultant residue, followed by extraction with diethylether. The organic phase was washed with water and aqueous saturatedbrine, and dried over anhydrous sodium sulfate. The solvent wasdistilled off under reduced pressure, and the resultant residue waspurified by vacuum distillation to thereby yieldN-t-butyloxycarbonyl-4-trimethylsilyloxy-1,2,3,6-tetrahydropyridine (32)(14.6 g) in the form of a colorless oil.

[0124] bp_(0.2) 107° C.

[0125] (2) Compound No. 32 (8.14 g) obtained in (1) and5,5-dimethoxy-1-pentyne (33) (4.23 g) were dissolved in absolutemethylene chloride (200 ml), and to the solution, a solution oftrimethylsilyltrifluoromethane sulfonate (270 μl) in absolute methylenechloride (15 ml) was added dropwise at −78° C. under argon gas, followedby stirring for 16 hours at the same temperature. Water (20 ml) wasadded thereto to thereby return the reaction mixture to roomtemperature, and the organic phase was removed. Subsequently, the waterphase was subjected to extraction with methylene chloride, and theextract was combined with the organic phase and washed with aqueoussaturated sodium hydrogencarbonate, water, and aqueous saturated brine.After the mixture was dried over anhydrous sodium sulfate, the solventwas distilled off under reduced pressure. The resultant residue waspurified by silica gel chromatography (n-hexane:ethyl acetate=5:1) tothereby yield N-t-butyloxycarbonyl-3-(1-mthoxy-4-pentinyl)-4-piperidone(34) (1.34 g) in the form of a colorless oil.

[0126] (3) Compound No. 34 (3.4 g) obtained in (2) was dissolved in amixed solvent of ethyl acetate (50 ml) and ethanol (50 ml), and sodiumborohydride (250 mg) was added to the solution, followed by stirring for30 minutes at room temperature. The reaction mixture was diluted withwater and subjected to extraction with ethyl acetate. The organic phasewas washed with water and aqueous saturated brine, dried over anhydroussodium sulfate, and concentrated under reduced pressure. The obtainedresidue was purified by silica gel column chromatography (n-hexane:ethylacetate=2:1) to thereby yieldN-t-butyloxycarbonyl-3-(1-methoxy-4-pentinyl)-4-piperidinol (35) (1.42g) in the form of a colorless oil.

[0127]¹H-NMR(CDCl₃) δ: 1.46(9H, s), 1.57-1.77(5H, m), 1.91-1.99(2H, m),2.23-2.37(3H, m), 3.14(2H, bs), 3.40(3H, s), 3.60(1H, bs), 3.94(1H, bs),4.17(1H, s)

[0128] (4) Compound No. 35 (1.16 g) obtained in (3) and4-(N,N-dimethylamino)pyridine (2.38 g) were dissolved in methylenechloride (50 ml), and acetyl chloride (0.55 ml) in methylene chloride (5ml) was added dropwise thereto under cooling on ice, followed bystirring for 2 hours at room temperature. After completion of thereaction, water was added thereto under cooling on ice, and 1 Nhydrochloric acid was added dropwise thereto for neutralization of themixture. Subsequently, the organic phase was removed, and the waterphase was subjected to extraction with methylene chloride. The extractwas combined with the organic phase, and the mixture was washed withwater and aqueous saturated brine. After the mixture was dried overanhydrous sodium sulfate, the solvent was distilled off under reducedpressure. The resultant residue was purified by silica gelchromatography (n-hexane:ethyl acetate=4:1) to thereby yieldN-t-butyloxycarbonyl-3-(1-methoxy-4-pentinyl)-4-acetoxypiperidine (36)(1.17 g) in the form of a colorless oil.

[0129] (5) Compound No. 36 (1.15 g) obtained in (4) was dissolved inchloroform (50 ml), and trifluoroacetic acid (7.70 ml) was addeddropwise to the solution under cooling on ice, followed by stirring for2 hours at room temperature. The reaction mixture was concentrated, andthe resultant residue was again dissolved in chloroform (100 ml). Afterthe solution was washed with aqueous saturated sodium hydrogencarbonateand the organic phase was dried over anhydrous sodium sulfate, thesolvent was distilled off under reduced pressure. The obtained residuein the form of a pale yellow oil was suspended in a mixed solvent ofacetonitrile (20 ml), water (10 ml), and methanol (20 ml). To thesuspension, 37 wt. % solution (1.50 ml) of formaldehyde was added andfurther, sodium cyanoborohydride (660 mg) was added thereto undercooling on ice, followed by stirring for 18 hours at room temperature.The reaction mixture was concentrated, and water (30 ml) was added tothe resultant residue. Acetic acid was added dropwise thereto to therebyadjust the pH of the mixture to 3, and then the resultant mixture wassubjected to extraction with chloroform. The organic phase was washedwith aqueous saturated sodium hydrogencarbonate and dried over anhydroussodium sulfate, and then the solvent was distilled off under reducedpressure. The obtained residue was purified by silica gel chromatography(methylene chloride:methanol=15:1) to thereby yieldN-methyl-3-(1-methoxy-4-pentinyl)-4-acetoxypiperidine (37) (340 mg) inthe form of a white amorphous powder.

[0130] (6) To a solution of compound No. 37 (300 mg) obtained in (5) andhydrogenated tri-n-butyltin (478 μl) in absolute toluene (10 ml),tetrakis(triphenylphosphine) palladium (68 mg) was added at 0° C. underargon gas, followed by stirring for 1 hour at room temperature. Thereaction mixture was concentrated, and the resultant residue wassubjected to silica gel chromatography (methylenechloride:methanol=20:1) to thereby obtain a fraction of interest. Thefraction was purified by NH silica gel chromatography (n-hexane:ethylacetate=2:1) to thereby yieldN-methyl-3-(1-methoxy-5-tributylstannyl-4-pentinyl)-4-acetoxypiperidine(38) (510 mg) in the form of a pale yellow oil.

[0131] The obtained compound No. 38 is a mixture of two differentgeometrical isomers, and the isomers can be separated into a firstisomer (38a) which elutes first and a second isomer (38b) which eluteslater, by means of elution with an eluent(methanol:water:triethylamine=90:10:0.2) by use of HPLC (ODS C18column). Each isomer has a different reactivity for AchE.

[0132] (7) Compound No. 38b (90 mg) obtained in (6) was dissolved inmethylene chloride (3 ml), and N-iodosuccinimide (45 mg) dissolved inmethylene chloride (5 ml) was gradually added dropwise to the mixture at0° C., followed by stirring for 5 minutes at the same temperature. Thesolvent was distilled off under reduced pressure, and the resultantresidue was purified by silica gel column chromatography (methylenechloride:methanol=10:1) to thereby yieldN-methyl-3-(1-methoxy-5-trans-iodo-4-pentinyl)-4-acetoxypiperidine (39)(41 mg) in the form of pale yellow needle-shaped crystals.

[0133]¹H-NMR(CDCl₃) δ: 1.44-1.53(1H, m), 1.63-1.81(2H, m), 1.86-1.96(2H,m), 2.08(3H, s), 2.09-2.17(4H, m), 2.32(3H, s), 2.62-2.65(1H, m),2.90-2.94(1H, m), 3.06-3.11 (1H, m), 3.29(3H, s), 5.02(1H, d), 6.02(1H,d),

Example 6

[0134] (1) Absolute methylene chloride (75 ml) was added to pyridiniumchlorochromate (2.61 g), and to the solution, a solution of the compoundNo. 17 (2.22 g) obtained in Example 2 (4) in absolute methylene chloride(25 ml) was added with stirring, followed by stirring for 3 hours.Insoluble matter was removed by filtration, and the filtrate wasevaporated to dryness. The resultant residue was purified by silica gelchromatography (n-hexane:ethyl acetate=1:1) to thereby yieldN-t-butyloxycarbonyl-3-methoxymethyloxy-4-formylpiperidine (40) (1.63 g)in the form of a colorless oil.

[0135] 1H-NMR(CDCl₃) δ: 1.46(9H, s), 1.48-1.59(3H, bs), 1.87(1H, d),2.51(1H, bs), 2.81-2.88(1H, bs), 3.36(3H, s), 3.78-3.84(1H, m), 3.96(1H,d), 4.65(1H, d), 4.74(1H, d), 9.75(1H, d)

[0136] (2) Absolute tetrahydrofuran (25 ml) was added totrimethylsilylacetylene (2.1 ml), and to the mixture, a solution (7.5ml) of 1.6 M n-butyllithium in hexane was added dropwise with stirringin the temperature range of −5° C. to 0° C. in an atmosphere of nitrogengas, followed by stirring for 10 minutes. The resultant mixture wasadded dropwise to a solution of compound No. 40 (1.63 g) obtained in (1)in absolute tetrahydrofuran (10 ml) with stirring in the temperaturerange of −5° C. to 0° C. in an atmosphere of nitrogen gas, followed bystirring for 1 hour and another round of stirring for 1 hour at roomtemperature. To the reaction mixture, a 10% aqueous solution (20 ml) ofammonium chloride was added, and the resultant mixture was diluted withwater and subjected to extraction with ethyl acetate. After the organicphase was washed with water and dried over anhydrous sodium sulfate, thesolvent was distilled off under reduced pressure. To the residue,methanol (30 ml) and an aqueous 5 N potassium hydroxide solution (3 ml)were added, and the resultant mixture was heated at 60° C. for 30minutes. The reaction mixture was concentrated, and to the resultantresidue, 10% aqueous solution (20 ml) of ammonium chloride was added.The resultant solution was diluted with water and subjected toextraction with ethyl acetate. The organic phase was washed with waterand dried over anhydrous sodium sulfate. The solvent was distilled offunder reduced pressure. To the residue were added methanol (30 ml) andan aqueous 5 N potassium hydroxide solution, and the mixture was heatedfor 30 minutes at 60° C. The reaction product was concentrated, and 10%aqueous ammonium chloride solution (20 ml) was added to the residue,followed by dilution with water and extraction with ethyl acetate. Theorganic phase was washed with water, dried over anhydrous sodiumsulfate, and purified by silica gel column chromatography(n-hexane:ethyl acetate=3:1) to thereby yieldN-t-butyloxycarbonyl-3-methoxymethyloxy-4-(l-hydroxypropargyl)piperidine(41). The obtained compound was separated into a first isomer (41a)which elutes first and a second isomer (41b) which elutes later, bymeans of elution with an eluent (hexane:2-propanol=100:3) by use ofChiral HPLC (CHIRALCEL 0J column). The yield of each isomer was 220 mg.FAB-MS (3-Nitrobenzyl alcohol) [M+H]⁺ 300

[0137]¹H-NMR(CDCl₃) δ: 1.46(9H, s), 1.50-1.59(2H, m), 1.75-1.84(2H, m),2.49(1H, d), 2.54(1H, bs), 2.64-2.67(1H, bs), 3.43(3H, s), 3.69-3.76(1H,m), 4.11-4.16(1H, bs), 4.59(1H, bs), 4.73-4.77(2H, dd)

[0138] (3) To compound No. 41b (50 mg) obtained in (2), absolutepyridine (500 μl) and acetic anhydride (250 μl) were added, and theresultant mixture was allowed to stand for 16 hours at room temperature.The reaction mixture was purified by silica gel chromatography(n-hexane:ethyl acetate=3:1) to thereby yieldN-t-butyloxycarbonyl-3-methoxymethyloxy-4-(1-acetoxypropargyl)piperidine(42) (56 mg) in the form of an oil.

[0139]¹H-NMR(CDCl₃) δ: 1.47(9H, s), 1.61(1H, bs), 1.78-1.84(1H, bs),2.01-2.06(1H, bs), 2.09(3H, s), 2.45(1H, d), 2.49-2.52(1H, bs),2.65-2.71(1H, bs), 3.34(3H, s), 3.37(1H, m), 4.13(1H, bs), 4.39(1H, bs),4.55(1H, d), 4.68(1H, d), 5.69(1H, d)

[0140] (4) To compound No. 42 (56 mg) obtained in (3), absolute toluene(3 ml), 2,2′-azobisisobutyronitrile (2 mg), and hydrogenated tributyltin(200 μl) were added, and the resultant mixture was stirred for 1 hour at85° C. in an atmosphere of nitrogen gas. The reaction mixture waspurified by silica gel chromatography (n-hexane:ethyl acetate=5:1) tothereby yieldN-t-butyloxycarbonyl-3-methoxymethyloxy-4-(1-acetoxy-3-tributylstannyl-2-propenyl)piperidine(43) (87 mg) in the form of an oil.

[0141]¹H-NMR(CDCl₃) δ: 0.88(15H, t), 1.25-1.34(6H, m), 1.43-1.52(7H, m),1.46(9H, s), 1.63-1.71(2H, bs), 2.09(3H, s), 2.55-2.65(2H, bs),3.31-3.35(1H, bs), 3.36(3H, s), 4.06(1H, bs), 4.38(1H, bs), 4.58(1H, d),4.70(1H, d), 5.64(1H, bs), 5.83(1H, dd), 6.08(1H, dd)

[0142] (5) Compound No. 43 (87 mg) obtained in (4) was dissolved inmethylene chloride (3 ml), and a solution of N-iodosuccinimide inmethylene chloride was added dropwise to the solution with stirringunder cooling on ice. At a point in time when the compound No. 43 haddisappeared, the solvent was distilled off under reduced pressure andthe resultant residue was purified by silica gel chromatography(n-hexane:ethyl acetate=5:1) to thereby yieldN-t-butyloxycarbonyl-3-methoxymethyloxy-4-(1-acetoxy-3-iodo-2-propenyl)piperidine(44) (48 mg) in the form of an oil.

[0143]¹H-NMR(CDCl₃) δ: 1.46(9H, s), 1.60-1.73(2H, m), 2.08(3H, s),2.52-2.66(2H, bs), 3.27-3.28(1H, bs), 3.35(3H, s), 4.08(1H, s), 4.55(1H,d), 4.68(1H, d), 5.60(1H, d), 6.40(1H, dd), 6.51(1H, d)

[0144] (6) To compound No. 44 (48 mg) obtained in (5), methylenechloride (3 ml) and trifluoroacetic acid (0.1 ml) were added, and theresultant mixture was allowed to stand for 16 hours at room temperature.The mixture was concentrated under reduced pressure, and to theresultant residue, methylene chloride (5 ml) was added. After one dropof concentrated ammonia water was added thereto with stirring undercooling on ice, the resultant mixture was dried over anhydrous sodiumsulfate and the solvent was distilled off under reduced pressure. To theresultant residue, acetone (5 ml) was added, and further, ¹⁴C-methyliodide (52 mCi/mmol) was added in an amount of 3 mCi, followed bystirring for 2 hours at room temperature. The solvent was distilled offunder reduced pressure, and methylene chloride (2 ml) andtrifluoroacetic acid (1 ml) were added to the resultant residue, and theresultant mixture was allowed to stand for 16 hours at room temperature.The solvent was distilled off, and methylene chloride (10 ml) and water(5 ml) were added to the resultant residue. After concentrated ammoniawater was added dropwise thereto with stirring under cooling on ice tothereby make the mixture alkaline, an organic phase was separated. Theorganic phase was washed with water, dried over anhydrous sodiumsulfate, and purified by silica gel chromatography(chloroform:methanol=15:1) to thereby obtainN-¹⁴C-methyl-3-hydroxy-4-(1-acetoxy-3-iodo-2-propenyl)piperidine (45)(12 mg, 1.8 mCi) in the form of a colorless oil.

[0145] FAB-MS(3-nitrobenzyl alcohol) [M+H]⁺ 340(¹²C), 342(¹⁴C)

[0146]¹H-NMR(CDCl₃) δ: 1.44-1.51(1H, m), 1.60-1.75(2H, m), 2.01-2.14(2H,m), 2.11(3H, s), 2.41(3H, s), 2.98(1H, d), 3.13-3.16(1H, m),3.53-3.58(1H, bs), 5.67(1H, d), 6.40(1H, d), 6.51(1H, dd)

[0147] (7) To compound No.45 (1.8 mCi, 12 mg) obtained in (6), methanol(5 ml) and 10% aqueous potassium hydroxide solution (0.125 ml) wereadded, followed by stirring for 2 hours at room temperature. The solventwas distilled off and water was added to the resultant residue, and withsalting out by an addition of sodium chloride, the resultant mixture wasextracted with methylene chloride. The extract was dried over anhydroussodium sulfate and concentrated to thereby obtain crudeN-¹⁴C-methyl-3-hydroxy-4-(l-hydroxy-3-iodo-2-propenyl)piperidine (46)(1.37 mCi).

[0148] FAB-MS(3-nitrobenzyl alcohol) [M+H]⁺ 298(¹²C), 300(¹⁴C)

[0149] (8) To compound No. 46 (1.37 mCi) obtained in (7), pyridine (0.4ml) and acetic anhydride (0.2 ml) were added, followed by stirring for 3hours at room temperature. The reaction mixture was purified by silicagel chromatography (chloroform:methanol=15:1) to thereby obtainN-¹⁴C-methyl-3-acetoxy-4-(1-hydroxy-3-iodo-2-propenyl)piperidine (47)(0.82 mCi, 6 mg) in the form of an oil.

[0150] FAB-MS(3-nitrobenzyl alcohol) [M+H]⁺ 340(¹²C), 342(¹⁴C)

[0151] The compound No. 47 was prepared as a ¹⁴C-labeled compound. When¹²C-methyl iodide was used as a raw material instead of ¹⁴C-methyliodide, non-radioactive compound No. 47 was obtained.

Example 7

[0152] In the same manner as described in Example 6, the followingcompounds were obtained.

Example 8

[0153] (1) Compound No. 11 (0.1 mg) obtained in Example 1 (9) wasdissolved in ethanol (50 μl), and ¹²³I-sodium iodide (74-185 MBq), 0.1 Nhydrochloride (50 μl), and 0.32% peracetic acid (50 μl) were added tothe solution, after which the resultant mixture was allowed to stand for30 minutes at room temperature with occasional stirring. To the reactionmixture, sodium metasulfite (50 μl) at a concentration of 100 mg/ml wasadded and further, a saturated solution of sodium carbonate (1 ml) wasadded. The resultant mixture was extracted with ethyl acetate. After theextract was dried over anhydrous sodium sulfate and concentrated underreduced pressure, the resultant residue was subjected to elution with aneluent (methanol:water:triethylamine=70:30:0.1) by use of HPLC (ODS C18column) to thereby obtain two geometrical isomers (50a) and (50b) ofradioactive iodide-labeled compound. Radiochemical purity of each isomerwas 98% or more. The geometrical isomers (50a) and (50b) have the sameretention time under HPLC and the same Rf value under TLC as do isomers(12a) and (12b) of non-radioactive compound No. 12.

[0154] (2) To geometrical isomer No. 50b obtained in (1),trifluoroacetic acid (0.5 ml) was added, and the resultant mixture wasallowed to stand for 2 hours at room temperature. The reaction mixturewas evaporated to dryness under reduced pressure, and the residue wassubjected to elution with an eluent(methanol:water:triethylamine=60:40:0.1) by use of HPLC (ODS C18 column)to thereby obtain radioactive iodide-labeled compound (51). The compound(51) has the same retention time under HPLC and the same Rf value underTLC as does a geometrical isomer (13b) of non-radioactive compound No.13.

[0155] (3) In the same manner as described in (1) and (2), the followingradioactive iodide-labeled compounds were obtained.

Test Example 1

[0156] (1) With respect to a geometrical isomer (50b) of compound No. 50obtained in Example 8 (1), reactivity and specificity for AchE wereexamined in accordance with the following method.

[0157] Cerebral cortical tissues were obtained from rats, weighed andhomogenized (90 mg tissue/ml) in 0.1 M phosphate buffer, pH7.4. To thehomogenate (200 μl), a solution (20 μl) of ¹²³I-labeled compound (50b)was added, and under incubation at 37° C. with time, hydrolysis rate wasmeasured by use of radio TLC. Meanwhile, when BW284c51, which is aspecific inhibitor for AchE, was added to the same reaction system,hydrolysis was considerably inhibited and specificity for AchE was88.6%.

[0158] (2) With respect to compound No. 13 led from an optical isomer(7b) of compound No. 7 obtained in Example 1 (5), reactivity andspecificity for AchE were examined in accordance with the followingmethod.

[0159] Rat brain cortex was processed into a 20% (w/v) homogenate by useof 0.9% NaCl-10 mM phosphate buffer (pH 7.4). To the homogenate, therewas added a ¹⁴C-labeled compound which had been prepared by substitutinga carbon atom of N-methyl group in compound No. 13 with ¹⁴C, and afterthe incubation at 37° C., hydrolysis rate was assayed by use of radioTLC. Two different components, one having a half-life of about 5 minutesand the other having a half-life of about 10 minutes, were identified.Through individual examination of two different geometrical isomers ofcompound No. 13, it was confirmed that the component having the shorterhalf-life corresponds to an isomer (13b) and the component having thelonger half-life corresponds to an isomer (13a). In contrast, whenBW284c51, which is a specific inhibitor for AchE, was added to the samereaction system, hydrolysis was considerably inhibited. From the resultsof the inhibition test, specificity for AchE in hydrolysis reaction ofisomers (13a) and (13b) were found to be 80.4% and 91.8%, respectively.Isomer (13b) exhibited excellent reactivity and specificity for AchE andit was confirmed that isomer (13b) has suitable characteristics forassaying the central AchE activity.

Test Example 2

[0160] After a geometrical isomer (50b) of compound No. 50 obtained inExample 8 (1) had been intravenously administered to groups of malewistar rats, radioactivity distribution in the rat brain was assayed bydissection method.

[0161] The results are shown in FIG. 1. Immediately afteradministration, radioactivity distribution in the rat brain depends onblood flow rate, which was found to be high in the brain cortex and lowin the striatum. Fifteen minutes or more after administration, highaccumulation of radioactivity was found in the striatum, which hadremarkably high AchE activity, but accumulation of radioactivity was lowin the cerebellum, which had low AchE activity. Therefore, radioactivitydistribution was found to vary in accordance with AchE activity.

Test Example 3

[0162] With respect to compound No. 13 induced from an chemical isomer(7b) of compound No. 7 obtained in Example 1 (5), a ¹⁴C-labeled compoundwhich had been prepared by substituting a carbon atom of the N-methylgroup in isomer (13b) with ¹⁴C was intravenously administered to a malewhite rat, and radioactivity distribution in the rat brain was assayedby use of quantitative autoradiography.

[0163] The results are shown in FIG. 2. Immediately afteradministration, radioactivity distribution in the rat brain depends onquantity of blood flow, which was found to be high in the brain cortexand low in the corpus striatum. Thirty minutes or more afteradministration, high accumulation of radioactivity was found in thestriatum, which had remarkably high AchE activity, but low accumulationof radioactivity was found in the cerebellum, which had low AchEactivity. Therefore, radioactivity distribution was found to vary inaccordance with AchE activity.

[0164] Tacrine (10 mg/kg), which is an inhibitor for central AchE, wasorally administered to a male white rat, and 30 minutes later, a¹⁴C-labeled compound which had been prepared by substituting a carbonatom of the N-methyl group in compound (13b) with ¹⁴C was intravenouslyadministered to the rat, and radioactivity distribution in the rat brainwas assayed by use of quantitative autoradiography. The distributionratio of radioactivity in each part of the brain was lowered by about30% as compared with the group to which Tacrine had not beenadministered.

[0165] The results show that central AchE activity relates closely todistribution of the present compound in the brain and suggest that thepresent compound is useful as a tracer for SPECT in assaying the centralAchE activity.

Test Example 4

[0166] Compound No. 47 obtained in Example 6 (8) and compound No. 46obtained in Example 6 (7), which is an acetylcholinesterase metaboliteof compound No. 47, were intravenously administered to a male white rat,and radioactivity distribution in the rat brain was assayed by use ofautoradiography.

[0167] The results are shown in FIG. 3. Compound No. 46, a metabolite,hardly migrated to the brain, but compound No. 47 commonly migrated tothe brain. Immediately (5 minutes later) after administration,radioactivity distribution in the rat brain depends on blood flow rate,and was found to be high in the brain cortex, somewhat high in thecorpus striatum, and low in the cerebellum. Thirty minutes afteradministration, high accumulation of radioactivity was found in thecorpus striatum, which had remarkably high AchE activity, but lowaccumulation of radioactivity was found in the cerebellum, which had lowAchE activity. Therefore, radioactivity distribution was found to dependon AchE activity.

[0168] The results show that central AchE activity relates closely todistribution of the present compound in the brain and that the presentcompound is useful as a tracer for SPECT in assaying the central AchEactivity.

[0169] Industrial Applicability

[0170] The compounds of the present invention have high lipophilicity,easily pass through the blood-brain barrier, are hydrolyzed specificallyby AchE within the central tissue into alcohols which have lowlipophilicity, which are then captured by the brain. In contrast,alcohols formed outside the brain do not migrate into the brain. Thecompounds of the present invention emit γ-rays at an appropriate energylevel. These characteristics make the compounds highly useful as tracersfor SPECT in assaying the central AchE activity.

1. An N-alkylpiperidine derivative represented by the following formula(1) or (2) or a salt thereof:

wherein R¹ represents an lower alkyl group which may be substituted by afluorine atom; R² represents a lower alkyl group; and R³represents analkenyl group which is substituted at the 1-position by a hydroxy group,a lower alkoxy group, a lower alkoxyalkyloxy group, a loweralkoxyalkyloxyalkyloxy group, or a lower alkanoyloxy group and issubstituted at an end by radioactive iodine, or an alkenyloxymethylgroup which is substituted at an end by radioactive iodine.
 2. AnN-alkylpiperidine derivative represented by the following formula (1P)or (2P) or a salt thereof:

wherein R¹ represents an lower alkyl group which may be substituted by afluorine atom; R² represents a lower alkyl group; and R^(3P) representsan alkenyl group which is substituted at the 1-position by a hydroxygroup, a lower alkoxy group, a lower alkoxyalkyloxy group, a loweralkoxyalkyloxyalkyloxy group, or a lower alkanoyloxy group and issubstituted at an end by a non-radioactive halogen atom, a trialkyltingroup, or a trialkylsilyl group, or an alkenyloxymethyl group which issubstituted at an end by a non-radioactive halogen atom, a trialkyltingroup, or a trialkylsilyl group.
 3. A reagent for assaying AchEactivity, which contains an N-alkylpiperidine derivative as described inclaim 1 or a salt thereof.
 4. A reagent for assaying central local AchEactivity according to claim 3, which is a reagent for use in singlephoton emission computed tomography (SPECT).
 5. An assay method forcentral local AchE activity, which comprises measuring radioactivityconcentration in a central local site by use of a reagent as describedin claim 3 or 4, measuring the blood flow in the central local site, andcomputing local AchE activity on the basis of a relation between theresultant measurements and AchE activity.